Tetracycline metallo-phosphate complexes and method preparation



Murray A. Kaplan, Syracuse, N. Y.

No Drawing. Application September 2-7, 1956, Serial No. 612,366

3 Claims. (Cl. 260-559) This invention relates to a new, therapeutically useful antibiotic compound and, more particularly, to tetracycline sodium hexametaphosphate complex.

It is the object of the present invention to provide a compound of tetracycline having improved therapeutic utility by comparison with the amp'hoteric tetracycline and the tetracycline hydrochloride which are now in clinical use; to provide such improvement it is necessary to increase the height or rate of attainment or duration of blood levels of tetracycline obtained" in man oranimals particularly upon oral administration of the tetracycline compound or salt.

The object of the present invention has been achieved and there is now provided by the present invention a tetracycline sodium hexametaphosphate compound.

More specifically, there is provided tetracycline sodium hexametaphosphate complex containing on an anhydrous basis the elements carbon, hydrogen, nitrogen, oxygen, sodium and phosphorus in a ratio substantially in accord with the empirical formula 5C22Hz4'N-z'Oa-NaPO3-SHPOs, which may also be described as therapeutically active, stable, non-toxic tetracycline'sodium hexametaphosphate complex characterized in purified form on an anhydrous basis by the presence of"0.7 to 1.1% sodium, 5.8 to 7.8% phosphorus, a potency in the range of 760 to 940 micrograms of tetracycline hydrochloride equivalents per milligram by bio-assay, and a solubility in Water at room temperature of about 3.2 mgm./ml. and further characterized by the production at the end of one and four hours after oral administration to man of a concentration in the blood stream substantially greater than that produced by the administration of an aquivalent amount of tetracycline hydrochloride.

The new tetracycline complex is preferably prepared by mixing aqueous, acid solutions of tetracycline, e. g. tetracycline hydrochloride, and sodium hexametaphosphate and collecting byfiltration under acid conditions the precipitated crystalline salt; The ratios by Weight of tetracycline hydrochloride to sodium hexametaphosphate to be used can vary widely; ratios of 1:2 to 1:005 are effective and about 1:0.25 or 1:0.33 is preferred. The acidity must be sufficient to maintain the tetracycline reagent in solution, e. g. less than about pH 2.0. The insolubility of the product makes the concentration used of little importance; reasonably concentrated solutions are, of course, more practical.

Tetracycline sodium hexametaphosphate complex gives higher blood levels in. dogs and in man upon oral administration in capsules than do the forms of tetracycline now in use, is non-toxic and is stable in the dry state.

The tetracycline sodium hexametaphosphate complex is very stable and can be used for oral application in powdered form, as'tablets or in capsules, but may also be used in suspensions in aqueous liquids or in anhydrous, edible oils, such as peanut oil, sesameoil' or a modified coconut oil with a setting point below 60 F.

When desired for specific purposes and rendered pharmaceutically compatible, there may-be admixedwith nited States Patent 0 ice a complex of the present invention various other addi tional medicaments, such as antihistamines, sulfa drugs, (e .g. sulfadiazine, sulfabenzamide, sulfacetamide, sulfanilamide, sulfapyridine, sulf'athia-zole, sulfapyrazine, sulfaguanidine, sulfathalidine, sulfasuxidine, sulfisoxazole, sulfamylon, phthalylsulfacetamide, N 3,4 dimethylbenzoylsulfanilamide, benzyl'sul fanilamide and N 2-(Z-quinoxalyl)sulfanilamide), lipotropic agents (particularly methionine, choline, inositol and beta-sitosterol and mixtures thereof), stimulantsof the central nervous system (e. g. caffeine, amphetamines), local anesthetics, analgesics (e. g. aspirin, salicylamide, sodium gentisate, p-acetylaminophenol, phenacetin, codeine), laxatives (e. g. phenolphthalein), sedatives (e. g.- barbiturates, bromides), salts of penicillin (e; g. potassium penicillin G, procaine penicillin G, l-ephenamine penicillin G, dibenzylamine penicillin G, other salts disclosed by U. S; Patent 2,627,491; these combinations are particularly useful to enable variation of the pattern of blood levels obtained), phenoxymethylpenicillin and salts thereof, other antibiotic agents (e. g. streptomycin, dihydrostreptomycin, bacitracin, polymixin, tyrothricin, erythro mycin, Aureomycin, Terramycin, oleandomycin, chloramphenicol, magnamycin, novobiocin, cycloserine; in some cases such combinations attack a Wider range of organisms or show synergistic efiicacy or provide decreased toxicity with'equal efficacy), vitamins (e. g. Vitamins A, A1, B1, B2, B6, B1: and members of that family, folic acid and members of that family, Vitamins C, D2, D3 and E), hormones (e. g. cortisone, hydrocortisone, 9a-fluorocorti-sone, 9ot-fluorohydrocortisone, prednisone and prednisolone), anabolic agents (e. g. 1l',1'7'-d'ihydroxy' 9a fiuoro 17oc-methyl-4-androsten-3-one; l7'-q ethyl-1'9 nortestosterone) and antifungal agents (e. g.', mycostatin) Analyses expressed herein as mcg./ml. or mcg/mgm. refer according to the usual custom to micrograms of tetracycline hydrochloride equivalents. Thus, pure tetracycline hydrochloride. has a potency of 1000 mcg./mgm. and pure tetracycline base of 1080 mcg./mgm., etc.

While the preferred embodiment of this invention is tetracycline sodium hexametaphosphate complex, a useful complex is also prepared by substituting potassium for the sodium of the preferred complex.

Average analytical values for nine to' twelve' separate preparations of tetracycline sodium hexametaphosphate complex was found to be as follows:

Moisture by vacuum-oven 9.7% (range 6.615.2%).,

technique. 7 Moisture by Karl Fischer 10.9% (range 8.4-16.7%).

Other values as corrected for moisture by Karl Fischer:

Phosphorus 6.8% (range 5.57.8%). Sodium 1.0% (range 08-11%). Potency in mcg./mgm. by 843 (range.760940).

bio-assay. Potency in mcg./mgm. by 843 (range 800-893).

ultra-violet absorption: Assay. I p

In the preparation of these samples, the ratio of='-the Weights of tetracycline hydrochloride to sodium hexametaphosphate used as reagents varied from 1:2 to 1:01 and was 1:033 in about half the cases; The difference between themoisture content as determined bythe vacuum-oven and Karl Fischer techniques indicates the presence of Water of crystallization, probably one" molecule per atom of sodium.

These results indicate that anhydrous tetracycline sodium hexametaphosphate complex has the empirical, and probably the molecular, formula 5C22H24N20a NaPOs SH-POi 3 for which the theoretical values are 0.85% sodium, 6.8% phosphorus and 880 mcg./mgm. potency, but this is purely a matter of theory and isnot essential to the present invention nor is the present invention restricted thereto. This empirical expression is so written for convenience and ease of comprehension and is particularly not intended to imply the existence in this product of any P03 ions, as the product is a complex hexametaphosphate. A more comprehensive understanding of this invention is obtained by reference to the following examples, which are illustrative only and are not the exclusive embodiment of the invention. 5

EXAMPLE 1 Weight Ratios of Sodium Hexametaphos- Yield in Bio-assay,

phate to Tetracycline Hydrochlorlde grams meg/mum.

This product gave blood levels in dogs of 1.29 and 1.80 meg/m1. respectively one and four hours after oral administration of a single dose of 12.5 mgm. tetracycline hydrochloride equivalents/kg. Under the same conditions, tetracycline hydrochloride levels are 0.77-0.92

meg/ml. after one hour and 0.75-0.76 meg/ml. after four hours. v

This product had a solubility in water of about 3.2 mgm./ ml. at room temperature.

EXAMPLE 2 Eighty grams of sodium hexametaphosphate (Calgon) was dissolved in about 1600 ml. water and the pH was adjusted to 1.5 with hydrochloric acid; this solution was then added with stirring to a solution of 240 g. tetracycline hydrochloride in 2.4 liters water (100 mgm./ml.). After some tetracycline sodium hexametaphosphate complex formed as a guru, the solution was decanted and seeded to produce crystalline tetracyline sodium hexametaphos phate complex (178 g.) after stirring two hours, collecting by filtration and washing thoroughly with water and then methanol. An additional 21 g. crystalline product was obtained from the gum by stirring it in water with seed crystals. The product gave the following analyses after correction for the presence of 8.4% water (Karl Fischer): Tetracycline hydrochloride equivalents in mcg./ V

mgm.: 763 (bio-assay); 815 (ultraviolet assay); sodium, 1.0%; phosphorus, 6.1%. The presence of one, or perhaps two, molecules of water of hydration was indicated by the value of 7.3% moisture found by the vacuum oven technique.

Theory for [5 tetracycline-NaPOs'SHPO3] is potency 880, 0.85% sodium and 6.8% phosphorus.

The complex gave blood levels in dogs of 1.87 and 1.08 meg/ml. respectively one and four hours after oral administration of a single dose of 12.5 mgm. tetracycline hydrochloride equivalents/ kg.

EXAMPLE 3 to 1.7 with concentrated hydrochloric acid and filtering. The first half of the solution was added rapidly; the mixture then grew turbid and the second half of the solution was added slowly over 30 minutes with seeding and stir ring. The yellow crystalline tetracycline sodium hexametaphosphate complex which precipitated was collected by filtration after stirring for three hours, washed with 1.5 I. water and then 1 1. methanol, dried 16 hours at 55 C. and found to weigh 1063 g. and 998 g. respectively. The two yields were combined and found to contain 10.7% moisture by Karl Fischer and, as corrected for moisture, 5.75% phosphorus and 1.1% sodium and to assay 803 and 860 megs. tetracycline hydrochloride equivalents/mgm. by bio-assay and ultraviolet absorption assay respectively.

This product gave blood levels in dogs of 1.74 and 1.30 meg/ml. respectively one and four hours after oral administration of a single dose of 12.5 iugrn. tetracycline hydrochloride equivalents/kg.

EXAMPLE 4 Five lots of tetracycline sodium hexametaphosphate complex were prepared by mixing aqueous solutions (adjusted to pH 1.5 with hydrochloric acid) of 25 g. tetracycline hydrochloride in 250 ml. water and sodium hexametaphosphate in the indicated amounts in 125 ml. water and collecting product having the following, tabulated properties:

Mots Product Potency Weight of true by Na P in sodium hexa- Yield Karl in Per- Permetaphosin g. Fischer by bioby ultracent cent pirate In g. percent assay violet assay 7 12. 5 940 893 0. 9 "a. 5 15. 5 l2. 7 840 800 7. 7 17. 5 1.0. 3 894 835 0 9 7. 8 17. 5 1D. 9 840 838 0 8 7. 7 7 10. 7 880 853 0 8 7. 0

1 Corrected for moisture.

EXAMPLE 5 Mois- Potency li un Yield in ture in Na in P in i c. g. percent percent percent by Karl by biogg Fischer assay Say 1 182 16.7 855 850 l. l 0. 7 2 I 13.6 855 843 1.0 1.0 4

EXAMPLE 6 Five gram lots of tetracycline hydrochloride in aqueous solution at 50 mgm./ml. were mixed with aqueous solutions of sodium hexametaphosphate (Calgon) adjusted to pH 1.5 with hydrochloric acid and containing 2.5 g., 1.66 g.,- 1.25 g. and 0.50 g. respectively of sodium hexametaphosphate. An immediate precipitate of tetracycline sodium hexametaphosphate complex precipitated either as crystals or as a guru which gradually crystallized. The yields after methanol-washing and drying over P205 were 3.25 g., 3.25 g., (bio-assay 660 mcg./mgm.), 2.75 g. and. 1..0 g. respectively... The product had a solubility :5 in water of about 3.1 mgm;/ml., ,gave blood 'levels in dogs of 2.59 and 1.54 meg/ml. respectively one and four hours after oral administration of a single dose of 12.5 mgm. tetracycline hydrochloride equivalents! kg. and "was found by analysis to contain 7 '.,0% phosphorus and 2.60% moisture.

EXAMPLE 7 Capsulescontaining the tetracycline sodium'hexametzu phosphate complex of the present invention were prepared by mixing the salt with 5% magnesium stearate (200 mesh) and as much lactose as needed for the size of capsule to be used, screening the mixture and filling into hard gelatin capsules. The amount of complex in each capsule was that equivalent to 250 mgms. tetracycline hydrochloride, .e. g. 340 mgms. EfOI a sample of complex having a potency by bio-assay of 730 mcg./mgm.

The blood levels produced in man upon oral administration of a single capsule containing the equivalent of .250 mgms. of tetracycline hydrochloride were determined for the above capsules and for two brands of tetracycline hydrochloride capsules usedas controls with the following results:

Blood levels in man Plasma at r the indicated number of :Numhours after administration Form of Tetracycline ber of Patients 7 1 2 3 4 8 24 hour hours hours hours hours hours Brand of of tetracycline hydrochloride '5-21' 0.55 1. 21 0. 87 0.70 0.16 Brand B-of tetracycline hydrochloride l022. 0. 41 0.92 0.99 0. 63 0.20 Tetracycline Sodium' Hexametaphosphate 1 Complex 9 0.94 1. 75v 1.22 0.21

It is obvious by inspection that the tetracycline sodium hexametaphosphate complex gave much higher blood levels Without loss of duration. An additional quantitative measure of this improvement is furnished in the usual way by plotting these blood levels and measuring (integrating) the areas under the curves. ,It is thus lfound that tetracycline sodium hexametaphosphate complex is absorbed nearly twice as efiiciently as tetracycline hydrochloride.

EXAMPLE 8 Tablets containing the following ingredients:

are prepared .by mixing all of the ingredients withthe exception of one-half of the magnesium stearate. The mixture is then slugged and granulated into 20 to 60 mesh granules. The remaining magnesium stearate is added to the granules, which are thoroughly mixed and finally tabletted.

t6 EXAMPLE :9

Aqueous oral suspensions are prepared by using the following formulations:

Kelmarpotassium alginate) .g Sucrose granulate I 647 Methyl ParabenU. .S. P.-(200 mesh 0:80 PropylParabenUpS. P. (200*mesh). 0.20

OherryFlavor Sodium Bisulfitc, reagent. Sodium Citrate U. S.'P Sodium Saccharin U. S. P. 2 mesh) Sodium Sucaryl (200 mesh) Tween 85 10 Sodium Metabisulflte g *1. 00 Gum Tragacantd g- 12.;00 Oil of Chocolate. .cc- .0. 7.5 011 oflPepperrnin 0. Sodium. Chloride g. ,3. 50 Distilled Water, 41. suto cc. 1, 0(10 1 000 Megs. oi Tetracycline per ml. oral) h xametaphos hate complex (200 mesh) is added under agitation, the volume made up to .1000 cc. with distilled water and the product filled into bottles.

Tetracycline sodium ,hexametaphosphate complex is distinguished from other tetracycline phosphates in many ways. The .most important :is the failure of the others to ,give blood levels higher than ;those obtained with -tetrac ycline hydrochloride; this has :been :found true .in particular of the tetracycline orthophosphate postulated .by-Conover-(UgS. Patent 2,699,054). iConover discloses only salts of strong mineral .;-acids, including phosphoric acid. Such an .orthorphosphate does not include a metallic ion, i. e. is not a tetracycline sodium ortho- -phosphate complex. :Specific illustrations of these differences from other tetracycline phosphates are given in the Comparison Examples below.

COMPARISON EXAMPLE-,1

Five gram lots of tetracycline hydrochloride in aqueous solution at 50 mgm./ml. were mixed with aqueous solutions containing 2.5 g., 1.66 g, 1.25 g., and 0.50 g. respectively of orthophosphoric acid (specific gravity 1.7; acid). The products Were adjusted to pH v1;5 if necessary. No solid precipitated either immediately or on standing.

COMPARISON EXAMPLE v2 COMPARISON EXAMPLE '3 Admixture of solutions adjusted to :pH 1.5 of :30 g. tetracycline hydrochloride in BOOmLWater andeither 13116 g. in 136 ml. Water or 10 g. in ml. water of NaH2PO4 H2O gave no precipitate either immediately or on standing.

COMPARISON EXAMPLE 4 An aqueous solution was prepared containing 5 g. tetracycline and 1.66 g. sodium orthophosphate (NasP04) in water adjusted to pH 1.5 and a volume of 50 ml. No solid precipitated.

Replacement of the sodium orthophosphate with sodium hexametaphosphate (Calgon) in a duplicate experiment precipitated 3.15 g. tetracycline sodium hexametaphosphate complex, bio-assaying 620 meg/mgm.

COMPARISON EXAMPLE 5 Five gram lots of tetracycline hydrochloride in aqueous solution at 50 mgm./ml. were mixed with aqueous solutions of sodium tetrametaphosphatc adjusted to pH 1.5 and containing 2.5 g., 1.66 g., 1.25 g. and 0.50 g. respectively of sodium tetrametaphospha-te. No solid precipitated either immediately or upon standing.

COMPARISON EXAMPLE 6 Five gram lots of tetracycline hydrochloride in aqueous solution at 50 mgm./ml. were mixed with aqueous solutions containing 2.5 g., 1.66 g., 1.25 g. and 0.50 g. re-

spectively of potassium polymetaphosphate which had been adjusted to pH 1.5 with hydrochloric acid. No substantial amount of solid precipitated except in the case where 0.50 g. potassium polymetaphosphate was meg/mgm. was obtained. This solid was soluble in water to the extent of about 2.0 mgm./ml. at room temperature.

COMPARISON EXAMPLE 7 Five gram lots of tetracycline hydrochloride in aqueous solution at 50 mgm./ml. were mixed with aqueous solutions of sodium hypophosphite adjusted to pH 1.5

and containing 2.5 g., 1.66 g., 1.25 g. and 0.50 g. re-

spectively of sodium hypophosphite. No solid precipitated either immediately or on standing.

COMPARISON EXAMPLE 8 Five gram lots of tetracycline hydrochloride in aqueous solution at 50 mgm./ml. were mixed with aqueous solutions of sodium tripolyphosphate adjusted to pH 1.5 with hydrochloric acid and containing 2.5 g. and 1.25 g. respectively of sodium tripolyphosphate. Solid precipitates formed in the amounts of 1.3 g. and 1.6 g. respectively and were combined, found to bio-assay 800 meg/mgm., to be soluble in water at room temperature 'to the extent of about 0.26 mgm./ml. and to give blood levels in dogs of 0.78 and 0.51 meg/ml. respectively one and four hours after oral administration of a single dose of 12.5 mgm. tetracycline hydrochloride equivalents/kg. Under the same conditions, tetracycline hydrochloride levels are 0.77-0.92 meg/ml. after one hour and 0.75- 0.76 meg/ml. after four hours.

COMPARISON EXAMPLE 9 Five gram lots of tetracycline hydrochloride in aqueous solution at 50 mgm./ml. were mixed with aqueous hydrochloride equivalents/ kg.

.used. In that case, 1.2 g. of solid bio-assaying 790 A 8 COMPARISON EXAMPLE 10 When a solution, adjusted to pH 1.5 of potassium meta phosphate is added to an aqueous solution of tetracycline hydrochloride at 5 0 mgm./ml, there is precipitated a solid, often gummy, which redissolves as additional amounts of metaphosphate are added. It was found that the use of 0.25 g. potassium metaphosphate per gram of tetracycline hydrochloride gave a gum which could be recrystallized from water and obtained as a yellow, crystalline solid, bio-assaying about 610 mcg./ml., giving blood levels in dogs of 0.72 and 0.45 meg/ml. respectively one and four hours after oral administration of a single dose of 12.5 mgm. tetracycline hydrochloride equivalents/kg. and exhibiting a solubility in water at room temperature of about 5.1 mgm./ml.

COMPARISON EXAMPLE 11 One gram of pure ammonium epitetracycline was dissolved in 10 ml. of water and adjusted to pH 1.8 by the addition of hydrochloric acid. Sodium hexametaphosphate (Calgon) (330 mgm.) was dissovlved in 5 ml. water and adjusted to pH 1.8 with hydrochloric acid. 'Upon mixing the two solutions, no precipitate was obtained, even upon seeding with tetracycline sodium hexametaphosphate complex and standing for three days.

COMPARISON EXAMPLE 12 In a cross-over test in seven patients, the following blood levels were obtained by single oral doses of amounts in capsules equivalent to 250 mgms. tetracycline hydrochloride:

Blood Levels ln meg/ml.- Hours after Administration Compound Tetracycline Sodium Hexametaphos- 1.58 2.68 2.61 1.35 0.29

phate Complex. Tetracycline Hydrochloride 0.83 1.37 1. 84 1.10 0.22

References Cited in the file of this patent UNITED STATES PATENTS OTHER REFERENCES Minieri et al.: Antibiotics Annual, 1953-54, pages 82 and 83. 

1. TETRACYCLINE METALLO-HEXAMETAPHOSPHATE COMPLEX WHEREIN SAID METALLO-HEXAMETAPHOSPHATE IS A MEMBER SELECTED FROM THE GROUP CONSISTING OF SODUIM HEXAMETAPHOSPHATE AND POTASSIUM HEXAMETAPHOSPHATE. 